Method of detecting malignant tumor and detection kit

ABSTRACT

A malignant tumor detection method suitable for the primary screening for a malignant tumor, and a detection kit using the method are provided. Considering various amino group-containing low molecular weight tumor markers in a sample, a tumor marker is labeled under suitable conditions using an amino group labeling dye which reacts specifically with it. The labeled tumor marker is subjected to spectroscopic measurement, and the tumor marker amount present in the sample is calculated. The presence or absence of a malignant tumor is detected by comparing the tumor marker amount with a threshold value for the tumor marker obtained from healthy persons. The series of these steps is performed using the detection kit.

TECHNICAL FIELD

This invention relates to a malignant tumor detection method anddetection kit, and in particular, relates to a simple malignant tumordetection method and detection kit using an amino group-containing lowmolecular weight tumor marker, in order to discover a malignant tumor atan early stage.

BACKGROUND ART

Examples of low molecular weight tumor markers for malignant tumorsknown in the art include amino group-containing compounds, such as3-hydroxyproline, pteridines and polyamine. Clinical research on suchamino group-containing compounds began to attract attention, afterRussell et al found in 1971 that polyamine excretion increased in cancerpatient urine (Russel, D. H. et al: Cancer Res., 31, 1555-1558 (1971)).

A widely used method for analyzing polyamine is the enzymatic method. Inthis method, the total amount of free polyamine (cadaverine, spermine,putrescine) is measured using acylpolyamine amidohydrolase. Using thismethod, many kinds of cancer, such as esophageal carcinoma, gastriccancer, colon cancer, cholangiocarcinoma, pancreatic cancer, lungcancer, ovarian cancer and prostatic cancer can be detected with asensitivity of close to 50% (Akiyuki Okubo, Medical Clinics of Japan,24, 11, 2234-2238 (1998)).

3-hydroxyproline is an amino acid specific to Type IV collagen which isa component of basal membrane. If cancer cells proliferate and basalmembrane is broken, 3-hydroxyproline is excreted in urine (Kubochi, K.et al: Development of a direct measurement assay for collagenase againstType I and Type IV collagens in tissue homogenate and its application instomach and lung cancers. In Proteinases in Inflammation and TumorInvasion (Tschesche, H. ed.), 337-356, Walter de Gruyter and Co.,Berline, 1986). Okazaki et al measured the 3-hydroxyproline amount inthe urine of 97 patients suffering from various types of cancer and 211healthy persons by the ninhydrin method using an automatic amino acidanalyzer. As a result, the sensitivity of 3-hydroxyproline was 42%. Onthe other hand, the percentage of healthy persons showing an abnormalvalue was only 2% (Okazaki, I. et al.: J. Lab. Clin. Med., 120, 908-920(1992)).

To detect and assay the various aforesaid amino group-containing lowmolecular weight tumor markers, liquid column chromatography has beenused conventionally. In liquid column chromatography, it takes long timeto establish the analysis conditions suitable for a specific marker.Further, separation procedure and detection also take long time, and thedetection reproducibility is not good. The chromatography unit itselfalso has the disadvantage of being difficult to miniaturize. Hence, itsclinical application to the diagnosis of a test subject was verydifficult.

DISCLOSURE OF THE INVENTION

It is therefore an object of this invention, which was conceived in viewof the present situation, to provide a simple and rapid malignant tumordetection method and detection kit using an amino group-containing lowmolecular weight tumor marker.

In order to attain the aforesaid object, the Inventor considered it apressing need to establish a method of simply detecting and assaying anamino group-containing low molecular weight tumor marker withoutdepending on liquid chromatography.

It was thus discovered that the amino group-containing low molecularweight tumor marker in a sample could be selectively labeled (even inthe presence of other amino group-containing low molecular weightcompounds in vivo) by an amino acid labeling dye, and that the amount ofthis labeled amino group-containing low molecular weight tumor markercould be assayed by a spectroscopic method. Further, by comparing thevalue of the amino group-containing low molecular weight tumor markeramount in the sample calculated in this way with a threshold for thistumor marker, a malignant tumor could be detected simply and rapidly,and this led to the present invention.

Specifically, the present invention provides a malignant tumor detectionmethod using an amino group-containing low molecular weight tumor markerin a sample, comprising the steps of:

-   -   (a) reacting the sample with an amino group labeling dye to        produce a labeled amino group-containing low molecular weight        tumor marker;    -   (b) measuring the fluorescence or absorbance of the sample after        the step (a);    -   (c) calculating the amount of the amino group-containing low        molecular weight tumor marker in the sample from the measured        value obtained in the step (b); and    -   (d) comparing the calculated value obtained in the step (c) with        the threshold value for the amino group-containing low molecular        weight tumor marker, where the result is deemed to be “positive”        for the presence of a malignant tumor if the calculated value is        larger than the threshold.

Unlike tumor markers such as antigens and antibodies, the low molecularweight tumor marker is nonspecific for tumor tissues, and the detectionmethod of this invention is suitable for the primary screening for amalignant tumor.

In the aforesaid malignant tumor detection method, a step of pretreatingthe sample prior to the step (a) may be included.

In the aforesaid malignant tumor detection method, a step of performinga concentration correction of the calculated value of the aminogroup-containing low molecular weight tumor marker may further beincluded between the steps (c) and (d).

In the aforesaid malignant tumor detection method, the amino grouplabeling dye is preferably 4-chloro-7-nitrobenzofurazan or4-fluoro-7-nitrobenzofurazan.

In the aforesaid malignant tumor detection method, the amino grouplabeling dye is preferably immobilized on a solid phase in advance.

In the aforesaid malignant tumor detection method, the step (b) ispreferably performed by fluorometry.

In the aforesaid malignant tumor detection method, the aminogroup-containing low molecular weight tumor marker is preferably3-hydroxyproline.

In the aforesaid malignant tumor detection method, the sample ispreferably urine.

In the aforesaid malignant tumor detection method, the concentrationcorrection is preferably performed by the creatinine amount in thesample.

The present invention further provides a malignant tumor detection kitusing an amino group-containing low molecular weight tumor marker in asample, comprising at least a standard solution of the aminogroup-containing low molecular weight tumor marker, an amino grouplabeling dye and a buffer.

In the aforesaid malignant tumor detection kit, the amino group labelingdye is preferably immobilized on a solid phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing measured values of the fluorescenceintensity of the reaction product of 3-hydroxyproline and4-chloro-7-nitrobenzofurazan (NBD-Cl) in urine specimens sampled fromhealthy persons and cancer patients according to the method of thepresent invention, wherein the measured values corrected by the creatineconcentration in the urine specimen for each sample have been plottedfor the healthy person group and the malignant tumor patient group.

BEST MODE FOR CARRYING OUT THE INVENTION

The malignant tumor detection method and detection kit according to thepresent invention will now be described in detail.

According to the invention, a malignant tumor is detected by selectivelylabeling an amino group-containing low molecular weight tumor marker inthe sample from a test subject, and assaying the labeled aminogroup-containing low molecular weight tumor marker.

(Amino Group-containing Low Molecular Weight Tumor Marker)

Tumor markers are substances which tumor cells produce in large amounts,and they are normally undetected substances which are also referred toas tumor-related antigens. Autoantibodies to these antigens are likewisegenerated only after the antigen is produced, which is why it can beused as a tumor marker. In addition, tumerigenic cells produce largeamounts of hormones, enzymes and specific low molecular weightcompounds, all of which may be useful to detect the presence of amalignant tumor (cancer).

The present invention utilizes an amino group-containing low molecularweight tumor marker, in particular, 3-hydroxyproline as a tumor marker.The reason for using an amino group-containing low molecular weighttumor marker, as was already mentioned, is that it permits easierdetection and assay than a macromolecule such as an antigen or antibody,and is suitable for the nonspecific detection of malignant tumors(tissues or organs) (i.e., primary screening).

(Amino Group Labeling Dye)

The present invention utilizes an amino group labeling dye for labelingan amino group-containing low molecular weight tumor marker, and whenthe marker is 3-hydroxyproline, 4-chloro-7-nitrobenzofurazan (NBD-Cl) or4-fluoro-7-nitrobenzofurazan (NBD-F) is preferably used as the aminogroup labeling dye, with NBD-Cl being more preferred.

(Sample)

The sample used in the invention may be any biological specimen usuallysampled in in vitro diagnosis, such as serum, plasma, urine, spinalfluid, amniotic fluid, ascites or lymphocytes, and rinses or extractsobtained by washing tissue or cells with solvents. Among these, urineand serum are preferable, and urine is particularly preferred from theviewpoint that it can be sampled without pain unlike collections ofblood.

(Pretreatment of Sample)

In order to react the sample with the amino group labeling dye, andlabel the amino group-containing low molecular weight tumor markercontained in the sample, it may be preferable to pretreat the samplebefore the labeling reaction. Although the pretreatment method used bythe invention varies with the sample type, a known method may be usedfor a specific sample. For example, if the sample is urine, the urinesampled from the test subject is centrifuged, and the supernatant ishydrolyzed by reaction with hydrochloric acid. Subsequently, thehydrolyzed urine supernatant is lyophilized, and reconstituted in abuffer. The buffer used for the reconstitution may be borate buffer,phosphate buffer or Tris buffer.

If necessary, the urine sample may be diluted with an organic solventmiscible with water. Examples of an organic solvent which may be usedfor this purpose are dimethylsulfoxide (DMSO), dimethylformamide (DMF)and ethanol. If the concentration of the labeled amino group-containinglow molecular weight tumor marker in the sample is too low forspectroscopy measurement (described later), it may be concentrated to anappropriate concentration. Specifically, the sample may be concentratedby removal of solvent, chromatography or the like.

(Labeling Reaction of Amino Group-containing Low Molecular Weight TumorMarker)

Subsequently, the sample is allowed to react with the amino grouplabeling dye, and the amino group-containing low molecular weight tumormarker in the sample is thereby labeled. In a particularly preferredform of the invention, if 3-hydroxyproline is selected as the aminogroup-containing low molecular weight tumor marker and NBD-Cl isselected as the amino group labeling dye, a classical reaction iscarried out as follows. The sample is brought in contact with NBD-Cl,and incubated at room temperature (about 20-50° C.) for a predeterminedtime (1 to 2 minutes). This reaction time is a sufficient time only forNBD-Cl and 3-hydroxyproline to react, and even in the presence of aminogroup-containing low molecular weight compounds (contaminant aminoacids, etc.) in the sample, as these have a low reactivity with NBD-Clunder the aforesaid reaction conditions, almost no reaction productswhich might interfere with the spectroscopic measurement are generated.

The catalytic reaction can be carried out if the reaction system is aliquid phase, or a liquid phase/solid phase system. In the former case,NBD-Cl is insoluble in water, and therefore is used in solution in anorganic solvent which is miscible with water. Specific examples of suchan organic solvent are methanol, ethanol, diethyl ether, ethyl acetate,DMF, DMSO, acetone and acetonitrile. Among these, acetonitrile isparticularly preferred.

The pH of the reaction system is preferably kept constant (particularly,within the range of approx. 8.0 to 9.0). For this purpose, a buffer isadded to the reaction system. Suitable buffers are borate buffer,phosphate buffer and Tris buffer.

(Immobilization)

When inspecting many samples at once using the malignant tumor detectionmethod of the invention, it is preferred to carry out the labelingreaction in a liquid phase/solid phase system.

For such purpose, the amino group labeling dye to be used is immobilizedon a solid phase. Examples of a suitable solid phase are a tube (glassor plastic) containing a microtube, a plate (for example, a 96/384 microwell plate), a pipette tip, polystyrene beads, latex particles ormagnetic particles. The material thereof and its shape are notparticularly limited provided that is a material which does notinterfere with the spectroscopy measurement of the sample after thelabeling reaction. The immobilization method used may be physicaladsorption, covalent bond or any other method known in the art. In thecase that the amino group labeling dye is immobilized on the solid phaseby physical adsorption, a surfactant and/or a dispersion adjuvant arepreferably used to disperse and immobilize the amino group labeling dyemore uniformly. Examples of a suitable dispersion adjuvant arepolyethylene glycol, cyclodextrin and dextran. Examples of a suitablesurfactant are Triton-X, sodium dodecyl sulfate (SDS) and Tween-20.

(Spectroscopic Measurement)

In the next step, the amino group-containing low molecular weight tumormarker labeled with the amino group labeling dye is measuredspectroscopically, and the amino group-containing low molecular weighttumor marker amount present in the sample is calculated. There is noparticular limitation on the spectroscopic method provided it canmeasure the fluorescence or absorbance of the labeling dye.

If 3-hydroxyproline is the low molecular weight tumor marker and NBD-Clis the amino group labeling dye, the labeled amino compound has afluorescence peak in the vicinity of about 550 nm, and an absorptionpeak in the vicinity of about 500 nm. Therefore, in this case, anapparatus which can measure the absorption spectrum of the visibleregion in the range of about 350 to 600 nm is desirable. An apparatuswhich allows the use of an excitation wavelength of about 250 to 550 nm,and which can measure the fluorescence spectrum in the range of about350 to 600 nm, is also desired.

When measuring the 3-hydroxyproline amount, it is more preferred fromthe viewpoint of detection sensitivity to measure the fluorescencespectrum (fluorescence intensity) of a fluorescent reaction product withNBD-Cl.

A calibration curve of the amino compound is constructed in advance byreacting a authentic sample of the amino compound, the aminogroup-containing low molecular weight tumor marker, with the amino-grouplabeling dye, measuring the value of the fluorescence intensity orabsorbance of the labeled amino compound, and plotting this against theconcentration of the amino compound used. The measured value of thefluorescence intensity or absorbance obtained from the sample is thencompared with this calibration curve, and the labeled aminogroup-containing low molecular weight tumor marker amount in the sampleis calculated.

(Concentration Correction)

When the sample is urine, it is necessary to make a concentrationcorrection of the calculated value of the amino group-containing lowmolecular weight tumor marker amount obtained above. This is because atest subject's water intake and water divergence differ from each other,and a dilution error rises in the calculated value. For this purpose,the creatine concentration in the sample is assayed, and the calculatedvalue is corrected based thereon.

(Detection of Malignant Tumor)

The malignant tumor is detected on the basis of the aminogroup-containing low molecular weight tumor marker amount in the samplewhich was calculated as described above. Herein, the aminogroup-containing low molecular weight tumor marker amount in the sampleof healthy persons is calculated in the same way as a comparison. Herealso, if the aforesaid concentration correction is necessary, it isperformed in a similar manner and a threshold for the aminogroup-containing low molecular weight tumor marker is obtained. Thisthreshold is compared with the calculated value (or corrected value) ofthe amino group-containing low molecular weight tumor marker in the testsubject's sample. As a result, if the calculated value is larger thanthis threshold, the test subject is deemed to be “positive” regardingthe presence of a malignant tumor, and a malignant tumor can thus bedetected. It should be stressed that the intention of the invention isto provide a means for primary screening of a malignant tumor. Apositive result means only that the test subject may suffer from amalignant tumor.

Preferably, a test subject who received such a positive result willproceed to inspections using other organ-specific diagnostic drugs (suchas CEA, AFP, PSA) or diagnostic methods, a biopsy, X-ray, MRI and thelike.

(Detectable Malignant Tumors)

The primary or metastatic malignant tumors which can be diagnosed usingthe detection method and detection kit of the invention are very varied,representative examples being breast cancer, prostatic cancer, livercancer, lung cancer, colorectal cancer, gastric cancer, pancreaticcancer, bladder cancer, head/neck cancer, kidney cancer, cervicalcancer, uterine cancer, thyroid cancer, brain tumors, tongue cancer,lymphoma, multiple myeloma, melanoma and leukemia. By using thedetection method and detection kit of the invention, these malignanttumors can be detected at various stages of progression, in particularat the first stage, and a discrimination can be made from the presenceof a benign tumor or the absence of a malignant tumor in the subjectbeing screened.

(Detection Kit)

In everyday diagnosis, a kit which does not require complicatedoperation is preferred so that a malignant tumor can be detected easily.As many physiologically active substances are present in vivo, adetection kit is required which can specifically discriminate and assaytumor markers. The invention provides a detection kit suitable for themalignant tumor detection method of the invention which satisfies thesevarious requirements, and comprises at least a standard solution of anamino group-containing low molecular weight tumor marker, an amino grouplabeling dye and a buffer. In addition to these components, it may alsocontain a sample diluent (solvent), solubilizing agent, solid phaseconversion standard reagent and positive control.

The buffer may be provided in the form of a solid or a liquid, such asborate buffer, phosphate buffer or Tris buffer generally used forbiochemical reactions, and a weakly alkaline buffer is preferred. Ifnecessary, as mentioned above, the buffer is used to maintain thelabeling reaction system of the amino group-containing low molecularweight tumor marker within a suitable pH range.

As described above, for the labeling reaction of the aminogroup-containing low molecular weight tumor marker, the reaction systemmay be a liquid phase or liquid phase/solid phase system.

Therefore, the detection kit can also take a corresponding form, thelatter system, i.e., a liquid phase/solid phase system is preferred asit maximizes the advantages of this invention. For this purpose, it ispreferred that the amino group labeling dye is immobilized on a solidphase. The detection kit having a construction wherein the amino grouplabeling dye is immobilized on a solid phase, enables measurement ofmany samples simultaneously, and detection can be easily performed. Asalready stated, it is particularly preferred that the solid phase usedis a microtube or a 96 micro well plate.

The form of the detection kit and container varies with the labelingreaction system (i.e., the combination of the amino group-containing lowmolecular weight tumor marker and amino group labeling dye), and the kitis not particularly limited if it comprises one set when measuring asample. One form thereof is given in the examples.

EXAMPLES

This invention will now be described in more detail, but this inventionis not to be construed as being limited in any way thereby.

Example 1 Assay of 3-hydroxyproline in Urine Specimen

(Preparation of Immobilized NBD-Cl)

100 μl of 10 mM NBD-Cl acetonitrile solution containing 0.01% ofTriton-X and 0.5% of polyethylene glycol were added into the wells of a96 well plate, and dried in a 30° C. oven for 2 hours.

The solid phase containing the immobilized NBD-Cl (96 well plate) can bestored at a cool and dark place if sealed under a nitrogen atmosphere.

(Pretreatment of Urine Specimen)

Urine sampled from healthy persons (21 samples) and malignant tumorpatients (17 samples) was centrifuged at 3000×g for 10 minutes. Thesupernatant of the urine obtained by centrifugation was collected, and 2ml of the each supernatant was introduced into a screw-top test tubecontaining 2 ml of 6M hydrochloric acid, and hydrolyzed at 150° C. for 2hours. 10 μl of the hydrolyzed urine was lyophilized, and 100 μl of 50mM borate buffer (pH 10.0) was added thereto.

(Labeling Reaction)

50 μl of the aforesaid treated urine was dispensed into the 96 wellplate immobilized with the aforesaid NBD-Cl, and reacted at roomtemperature for 1 minute with stirring. 100 μl of 0.5M HCl was thenadded thereto.

(Fluorometry)

For the sample in each well, the fluorescence intensity was measuredusing a fluorescence plate reader under the conditions that excitationwavelength is 505 nm and fluorescence wavelength is 560 nm. In order tocorrect the dilution error based on the water intake (and/or waterdivergence) of-each person, creatine in the same urine was measured by acreatine test Wako kit (Wako Pure Chemicals), and the 3-hydroxyprolineamount in the urine was calculated as a 3-hydroxyproline value per 1 gcreatine in the urine (mg/g creatine). FIG. 1 is a plot of thus obtainedcorrected values.

As shown in FIG. 1, from the fluorometry result of the urine sampledfrom the malignant tumor patients and the urine extracted from thehealthy persons, a difference was observed in the fluorescence intensityvalue (corrected). Specifically, the measured values for the malignanttumor patients were significantly larger than the measured values forthe healthy persons. The average of the healthy persons' measured valueswas taken, and using this as the threshold value (0.3), the tumorpatients' measured values were all larger than the threshold valueexcept for one example (among 17 samples). This test result shows that,apart from one person, the comparison of the fluorescence intensityvalues measured for all the test subjects, correspond with the clinicaldiagnosis regarding the presence or absence of malignant tumors in thesesubjects. Therefore, a malignant tumor can be detected using themalignant tumor detection method and detection kit of the presentinvention.

Industrial Applicability

As shown above, according to the malignant tumor detection method of thepresent invention, a malignant tumor can be rapidly and simply detectedby calculating the amino group-containing low molecular weight tumormarker amount in the sample, and comparing the calculated value with athreshold value.

Further, the detection of various amino group-containing low molecularweight tumor markers which was previously impossible, can now beperformed by means of samples used in general laboratory tests, such asserum and urine.

The malignant tumor detection kit of the present invention is suitablefor the application of the method of the present invention, and can bestored for long periods to allow the application of the method of thepresent invention when required.

According to the preferred examples of the malignant tumor detectionmethod and detection kit of the present invention, the amino labelingdye is immobilized on a solid phase, and as the amino group-containinglow molecular weight tumor marker is produced when a sample is added tothe solid phase, a malignant tumor can be detected more rapidly.Further, the detection kit can be handled more easily, and permitsdetection of a large number of samples at the same time.

1. A malignant tumor detection method using an amino group-containinglow molecular weight tumor marker in a sample, comprising the steps of:(a) reacting the sample with an amino group labeling dye to produce alabeled amino group-containing low molecular weight tumor marker; (b)measuring the fluorescence or absorbance of the sample after the step(a); (c) calculating the amount of the amino group-containing lowmolecular weight tumor marker in the sample from the measured valueobtained in the step (b); and (d) comparing the calculated valueobtained in the step (c) with the threshold value for the aminogroup-containing low molecular weight tumor marker, where the result isdeemed to be “positive” for the presence of a malignant tumor if thecalculated value is larger than the threshold.
 2. The malignant tumordetection method according to claim 1, wherein said amino group labelingdye is immobilized on a solid phase in advance.
 3. The malignant tumordetection method according to claim 1, further comprising a step ofpretreating the sample prior to the step (a).
 4. The malignant tumordetection method according to claim 3, further comprising a step ofperforming a concentration correction of the calculated value of theamino group-containing low molecular weight tumor marker between thesteps (c) and (d)
 5. The malignant tumor detection method according toclaim 4, wherein the amino group-containing low molecular weight tumormarker is 3-hydroxyproline.
 6. The malignant tumor detection methodaccording to claim 5, wherein the amino group labeling dye is4-chloro-7-nitrobenzofurazan or 4-fluoro-7-nitrobenzofurazan.
 7. Themalignant tumor detection method according to claim 6, wherein the step(b) is performed by fluorometry.
 8. The malignant tumor detection methodaccording to claim 7, wherein the amino group labeling dye isimmobilized on a solid phase in advance.
 9. The malignant tumordetection method according to claim 2, wherein the sample is urine. 10.The malignant tumor detection method according to claim 9, furthercomprising a step of performing a concentration correction of thecalculated value of the amino group-containing low molecular weighttumor marker between the steps (c) and (d).
 11. The malignant tumordetection method according to claim 10, wherein the concentrationcorrection is performed by the creatinine amount in the sample.
 12. Amalignant tumor detection kit using an amino group-containing lowmolecular weight tumor marker in a sample, comprising at least astandard solution of the amino group-containing low molecular weighttumor marker, an amino group labeling dye and a buffer.
 13. Themalignant tumor detection kit according to claim 12, wherein said aminogroup labeling dye is immobilized on a solid phase.